The  Normal  Mode  of  Secretion  in 
the  Thyroid  (Hand 


R,  R.  BENSLEY 

From  tlif  Hull  Laboratory  of  Anatomy,  University 
of  Chicago 


Reprinted  from  THE  AMERICAN  JOURNAL  OF  ANATOMY, 
Vol.  19.  No.  1,  January.  1916 


Reprinted  from  THE  AMERICAN  JOURNAL  OF  ANATOMY,  VOL.  19,  No.  1 
January,  1916 


THE  NORMAL  MODE  OF  SECRETION  IN  THE 
THYROID  GLAND 


R.  R.  , 

From  the  Hull  Laboratory  of  Anatomy, '  University  of  Chicago 

ONE    PLATE    IN   COLOR 

In  the  glands  of  the  alimentary  canal  the  process  of  secretion  is 
associated  with  definite  changes  in  the  structure  of  the  secreting 
cells,  and  with  the  accumulation  in  them  of  products,  granular 
or  otherwise,  which  may  be  interpreted  as  the  organic  antece- 
dents of  the  secretion  itself.  Even  in  some  of  the  internal  secret- 
ing glands,  as,  for  example,  the  islets  of  Langerhans  of  the  pan- 
creas, functioning  is  associated  with  the  storage  or  exhaustion  of 
intracellular  products  which  may  be  similarly  interpreted.  By 
means  of  these  secretion  antecedents  an  observer,  who  has,  by 
experiment  and  observation,  acquainted  himself  with  the  secre- 
tory mode,  may  form  an  estimate  of  the  secretory  potential  at 
the  time  of  observation. 

In  the  thyroid  gland,  on  the  other  hand,  the  search  for  such 
evidences  of  secretory  activity,  has  been,  as  regards  the  nature 
of  the  intracellular  secretion  antecedents,  of  so  contradictory  a 
nature,  and  of  such  doubtful  functional  import,  that,  at  present, 
we  are  unable  to  state  from  the  examination  of  a  thyroid  gland 
whether  the  gland  was  active  or  inactive.  Accordingly,  differ- 
ent observers,  as,  for  example,  in  Grave's  disease,  in  discuss- 
ing the  same  results,  have  arrived  at  diametrically  opposed 
conclusions. 

One  of  the  features  of  the  thyroid  gland,  in  particular,  which 
baffled  interpretation  was  the  presence  in  it  of  a  storage  product, 
the  so-called  colloid,  the  route  and  rate  of  resorption  of  which 
have  remained  problematical,  though  chemical  and  physiologi- 
cal studies  indicated  that  it  contained  the  physiologically  active 

37 

THE  AMERICAN  JOURNAL  OF  ANATOMY,  VOL.  19,  NO.  1 


'37091 


38  R.    R.    BENSLEY 

thyroid  substances.  Some  observers  have  even  doubted  the 
resorption  of  this  material,  and  have  suggested  that  the  function 
of  the  thyroid  gland  was  primarily  to  withdraw  toxic  substances 
from  the  blood.  Others  have  conceived  the  colloid  as  a  sort  of 
menstruum  in  which  the  real  thyroid  secretion  was  received  and 
from  whidji.'it  might  bfc -withdrawn  without  visible  change  in  the 
colloid  itself.  ..Siifl  others'  h.ave  held  the  view  that  the  colloid 
was  tbe*.]jekl:  ^rgti^n  of  ,-the  Jhyroid  gland  and  that  the  normal 
mechanism  of  thyroid  secretion  was  by  this  indirect  route,  first 
secreting  into  the  centre  of  the  follicle,  and  then  withdrawing 
this  ware-housed  material,  as  functional  needs  required,  by  some 
unknown  method  and  route. 

The  determination  of  the  true  significance  of  the  colloid  in  the 
secretory  cycle  of  the  gland,  and  of  the  ways  in  which  it  is  formed, 
and  of  its  intracellular  antecedents,  is  of  fundamental  importance 
in  the  physiology  and  pathology  of  the  thyroid  gland.  The  con- 
viction that  it  is  by  this  indirect  method  that  the  thyroid  gland 
produces  its  internal  secretion  lies  at  the  bottom  of  all  of  our 
more  or  less  speculative  interpretations  of  pathological  conditions, 
and  in  view  of  the  strong  physiological  evidence  supporting  this 
conviction  few  have  had  the  courage  to  question  its  accuracy. 
Many  authors  have  tried  nevertheless  to  influence  experimentally 
the  rate  of  secretion  in  the  gland,  and  to  read  in  the  changes  so 
produced  the  true  history  of  its  secretory  process.  In  this  way 
many  interesting  facts  have  been  discovered,  which  at  present 
seem  to  some  extent  contradictory  of  one  another,  but  which 
nevertheless  must  be  found  to  be  in  accord  when  the  true  history 
of  the  process  is  revealed. 

Our  earliest  knowledge  as  to  the  origin  of  the  intrafollicular 
colloid  of  the '  thyroid  gland  is  due  to  Biondi  and  Langendorff. 
Biondi  ('89)  showed,  that  this  substance  was  a  true  product  of 
the  secretory  activity  of  the  thyroid  epithelial  cells,  inasmuch 
as  he  found  globules  of  similarly  staining  substances  in  the  cells 
themselves.  He  conceived  the  process  of  secretion  as  follows: 
the  cells  of  the  thyroid  gland  produce  the  colloid,  .since  one  can 
see  in  them  little  globules  having  the  same  microchemical  reac- 
tions; the  vesicle  has  a  tendency  to  increase  in  size  partly  by 


NORMAL  MODE   OF   SECRETION  IN  THYROID   GLAND  39 

multiplication  of  the  epithelial  cells,  partly  by  increase  of  the  col- 
loid; after  filling  itself  the  vesicle  discharges  into  the  nearest 
lymphatic  vessel;  finally  the  collapsed  vesicle  disposes  itself  in  the 
form  of  a  number  of  little  acini  which  repeat  the  process. 

Langendorff  ('89)  using  the  method  of  comparative  study  for 
the  elucidation  of  the  secretory  process  in  the  cells  of  the  thy- 
roid gland,  reached  conclusions  which,  in  some  respects,  confirm 
and  extend  those  of  Biondi.  He  described  two  sorts  of  cells  in 
the  gland  which  he  designated,  respectively,  principal  cells,  and 
colloid  cells.  The  principal  cells  constituted  the  main  mass  of 
the  epithelium.  They  were  cylindrical  or  columnar  cells,  of 
variable  height  in  different  species  and  in  different  ages  of  the 
same  animal  species.  They  possessed  a  reticular  protoplasm, 
with  granules  at  the  nodal  points,  and  an  oval  or  round  nucleus 
situated  at  the  basal  end  of  the  cell.  Like  Biondi  he  saw  occa- 
sionally in  these  cells  small  hyaline  spherules,  but  considered 
them  to  occur  very  rarely.  The  colloid  cells  differed  from  the 
principal  cells  by  the  hyaline,  transparent  appearance  of  their 
cytoplasm.  This  cytoplasm  browned  with  osmic  acid,  and,  in 
dyes,  stained  the  same  as  the  colloid  content  of  the  follicles. 
He  found  all  grades  of  transition  between  the  colloid  cells  and  the 
principal  cells.  He  regarded  the  colloid  cells  as  elements  en- 
gaged in  the  secretion  of  colloid  but  did  not  commit  himself 
definitely  to  the  opinion  that,  after  a  period  of  secretion,  they 
might  return  to  the  state  of  the  principal  cells.  He  was  like- 
wise in  doubt  whether  they  degenerated  or  not  after  secretion. 

V.  Wyss  ('89)  studied  the  effects  on  the  thyroid  gland  pro- 
duced by  poisoning  with  pilocarpine.  He  found  in  cats  and  dogs 
that  the  gland  after  pilocarpine  was  large,  turgid,  and  filled 
with  blood,  and  that  the  cells  were  larger,  the  nuclei  less  appar- 
ent. The  free  ends  of  the  cells  were  prolonged  into  processes 
which  were  continuous  with  the  colloid  mass,  and  between  these 
processes  were  brilliant  spherules  of  apparently  fluid  nature. 

Anderson  ('94)  confirmed  V.  Wyss'  conclusions  relative  to  the 
effect  of  pilocarpine  on  the  gland,  and  studied  the  structure  of 
the  epithelial  cells  in  young  cats  and  rabbits  at  different  periods 
of  time  after  injections  of  pilocarpine.  He  described,  in  the 


40  R.    R.    BENSLEY 

earlier  phases  of  pilocarpinisation,  the  appearance  of  clear  drop- 
lets in  the  cytoplasm,  which  collected  at  the  free  pole  of  the 
cell,  to  be  extruded  in  the  form  of  small  droplets  into  the  cavity 
of  the  vesicle.  These,  therefore,  he  regarded  as  the  antece- 
dents of  the  clear  vacuoles  of  the  margin  of  the  colloid  and  on 
account  of  their  lack  of  affinity  for  dyes,  designated  chromo- 
phobe  secretion.  A  little  later  round,  stainable  droplets  made 
their  appearance  in  the  free  pole  of  the  cell,  which  likewise  mi- 
grated to  the  free  border  to  be  extruded  into  the  lumen,  consti- 
tuting thus  the  chromophile  secretion.  He  regarded  the  colloid 
cells  of  Langendorff  as  cells  destined  to  degenerate.  Thus, 
Anderson  rejected  the  mode  of  secretion  favored  by  Langendorff, 
and  introduced  the  conception  of  a  polyvalent  secretion.  His  re- 
sults, in  general,  are  in  more  accord  with  those  of  Biondi  than 
with  those  of  Langendorff. 

Htirthle  ('94)  in  the  same  year  studied  the  effects  of  reduc- 
tion of  thyroid  tissue,  and  of  bile  retention,  on  the  secretory  proc- 
esses of  the  gland.  He  found,  as  a  result  of  each  of  these  con- 
ditions, a  great  increase  in  the  number  of  cells  containing  colloid 
spherules,  which  -he  therefore  interpreted  as  an  evidence  of  accel- 
erated activity  of  the  gland.  On  the  other  hand  he  recognized  the 
occurrence  of  the  Langendorff  colloid  cells  to  which  he  also 
ascribed  secretory  significance,  and  which  he  considered  capable 
of  transformation  into  principal  cells. 

Galeotti  ('96)  studied  the  thyroid  glands  of  the  turtle  Emys 
europaea,  under  normal  conditions,  and  after  the  injection  of 
various  products  of  metabolism.  He  described  two  sorts  of  secre- 
tion antecedents:  fuchsinophile  granules  of  nuclear  origin,  pre- 
viously undescribed,  and  droplets  of  colloid  like  those  described 
by  Biondi,  Anderson,  and  Hurthle.  These  two  secretion  ante- 
cedents varied  independently  of  one  another  under  the  experi- 
mental conditions  employed. 

Following  Galeotti  a  number  of  different  observers  using  his 
methods  studied  the  thyroid  gland  under  different  experimental 
and  pathological  conditions,  confirming  his  results  as  to  the 
double  character  of  the  thyroid  secretion  and  the  independent 
variation  of  the  two  sorts  of  secretion.  Among  these  may[be 


NORMAL  MODE  OF  SECRETION  IN  THYROID  GLAND      41 

mentioned  Tiberti,  and  Ciulla.  The  latter  identifies  the  fuchsin- 
ophile  granulations  of  Galeotti  with  the  chromophobe  secretion 
of  Anderson,  the  plasmosomes  with  the  chromophile  secretion  of 
the  same  author. 

Lobenhoffer  ('09)  studied  human  thyroid  glands,  both  normal 
and  pathological,  in  material  fixed  in  formol  Mtiller,  and  stained 
in  anilin  acid  fuchsin.  He  found,  in  his  preparations,  the  cells 
containing  in  widely  varying  amounts  spherical  fuchsinophile 
granules  about  the  size  of  the  granules  of  eosinophile  leucocytes. 
Sometimes  these  granules  formed  a  narrow  row  along  the  margin 
of  the  cell,  and  sometimes  very  small  granules  were  found  actu- 
ally in  the  margin  of  the  colloid.  These  granules  he  regarded  as 
the  antecedents  of  the  thyroid  secretion,  interpreting  the  vary- 
ing contents  as  indicating  different  phases  of  secretory  activity. 

As  a  result  of  the  work  of  these  observers  we  have  to  consider 
the  following  structures,  in  connection  with  the  secretory  ac- 
tivity of  the  thyroid  cells,  as  possible  intracellular  secretion 
antecedents : 

1.  Spherules   of   colloid,    described   by   all   observers   except 
Lobenhoffer. 

2.  Vacuoles  containing  a  colorless  unstaining  fluid  substance 
described  by  Anderson  as  chromophobe  secretion,  and  inter- 
preted by  him  as  the  antecedent  of  the  content  of  the  vacuoles 
seen  in  the  margin  of  the  colloid. 

3.  Colloid  occurring  as  a  diffusely  distributed  substance  in  the 
cytoplasm  of  the  so-called  colloid  cells  of  Langendorff. 

4.  The  fuchsinophile  granules  of  Galeotti. 

5.  The  fuchsinophile  granules  of  Lobenhoffer. 

Recent  work  by  O.  Schultze,  and  Mawas  has  helped  to  reduce 
the  number  of  supposed  secretion  antecedents  in  the  preceding 
list  by  demonstrating  the  presence  in  the  thyroid  epithelial  cells 
of  numerous  mitochondria,  usually  filamentous,  and  oriented  in 
the  direction  of  the  main  axis  of  the  cells.  There  seems  to  be 
little  doubt  that  the  fuchsinophile  granules  of  Lobenhoffer  are  in 
reality  mitochondria,  rather  imperfectly  preserved.  To  this  cate- 
gory belong  also  in  part  at  least  the  fuchsinophile  granules  of 
Galeotti,  Tiberti,  and  others.  Possibly  however,  a  part  of  these 


42  R.    R.    BENSLEY 

granule's  are  of  another  nature,  since  I  have  shown  that  in  hyper- 
plastic  glands  of  the  opposum  and  in  human  glands  from  cases 
of  exophthalmic  goiter,  non-mitochondrial  fuchsinophile  granules 
occur.  These  will  require  further  discussion  when  the  secretory 
by-products  are  considered. 

With  the  exception  of  Hurthle,  Langendorff ,  and  Schmidt,  prac- 
tically all  observers  agree  that  the  colloid  cells  of  Langendorff 
are  cells  in  the  last  stages  of  cytomorphosis.  The  perfect  grada- 
tion between  these  cells  and  the  so-called  principal  cells  on  the 
one  hand  and  the  obviously  degenerating  cells  of  the  follicle  on 
the  other  hand  leaves  little  doubt  of  their  significance.  The 
changes  in  the  nucleus,  the  disappearance  of  mitochondria,  and, 
in  many  cases,  the  visible  disintegration  of  the  cytoplasm,  or 
desquamation  of  the  cell,  all  point  to  the  correctness  of  this 
conclusion. 

Thus  by  elimination  we  arrive  at  the  conclusion  that  the  only 
secretory  antecedents  thus  far  demonstrated  in  the  thyroid  epi- 
thelial cells  which  may  be  considered  to  be  normal  products,  are 
the  colloid  globules  of  Biondi,  and  Hurthle,  to  which  belong  also 
the  chromophile  granules  of  Anderson,  and  the  so-called  chromo- 
phobe  secretion  of  Anderson.  It  is  necessary,  therefore  to  exam- 
ine in  greater  detail  the  occurrence  of  these  products  in  the  thy- 
roid epithelial  cells,  with  the  object  of  determining  whether  they 
are  actually  related  to  the  formation  of  intrafollicular  colloid, 
whether  they  are  sufficient  to  account  for  the  physiological  ac- 
tivity of  the  gland,  and  what  indications  they  afford  of  the  rate 
of  formation  of  the  intrafollicular  colloid. 

Hurthle  found  that,  when  the  thyroid  tissue  was  reduced  by 
the  removal  of  the  whole  of  one  lobe  and  two-thirds  of  the  other, 
in  many  places  in  the  gland  the  epithelial  cells  contained  droplets 
of  substance  which  was  sharply  defined  by  its  staining  reaction 
from  the  surrounding  protoplasm,  but  agreed  in  all  respects  with 
the  colloid  contained  in  the  follicular  lumina.  Similar  results 
he  obtained  by  ligating  the  common  bile  duct  and  the  thoracic 
duct  simultaneously.  In  these  glands  also  he  found  the  lym- 
phatic vessels  much  dilated  and  filled  with  strongly  staining  col- 
loid substance  without  admixture  of  formed  elements.  He  con- 


NORMAL  MODE  OF  SECRETION  IN  THYROID  GLAND  43 

sidered  two  possible  explanations  of  this  phenomenon,  namely, 
that  it  was  due  to  lymphatic  obstruction,  and  that  it  was  due  to 
accelerated  activity  of  the  gland,  and  decided  in  favor  of  the  latter 
alternative  because  ligation  of  the  thoracic  duct  alone  produced  no 
such  changes  in  the  gland. 

Langendorff  (loc.  cit.)  on  the  other  hand,  while  admitting  the 
occasional  occurrence  of  colloid  droplets  in  the  cells,  did  not  con- 
sider them  of  much  significance  from  the  secretory  standpoint 
because  of  their  extreme  rarity.  Anderson  also  saw  them,  not 
in  the  normal  cell,  but  as  a  result  of  prolonged  pilocarpinisation 
of  the  animal.  Schmidt  could  find  no  effect  on  the  structure 
of  the  epithelial  cells  as  a  result  of  pilocarpine  injections,  and  at- 
tached more  importance  to  the  colloid  cells  as  an  indication  of 
secretory  activity.  Bensley  ('14)  on  the  contrary,  in  studying 
the  involution  of  the  hyperplastic  gland  of  the  opossum  pro- 
duced by  the  administration  of  iodides,  found  that  the  cells  of 
the  gland  practically  all  contained  globules  of  colloid,  and  that 
they  could  be  seen  discharging  it  into  the  lumen,  while  colloid 
cells  were  almost  completely  lacking.  In  this  case  the  restoration 
of  the  intrafollicular  colloid  was  wholly  by  the  formation  of  in- 
tracellular  globules  which  discharged  into  the  lumen.  The  proc- 
ess however  was  an  extremely  slow  one;  after  seventeen  days, 
though  practically  every  cell  contained  a  globule  of  colloid,  as 
large  as,  or  larger  than  the  nucleus,  there  was  little  intrafollicular 
colloid,  and  at  the  end  of  twenty-four  days  of  daily  administra- 
tion of  iodides  the  condition  was  but  slightly  advanced;  intra- 
cellular  colloid  remained  about  the  same  as  in  the  preceding  case 
but  the  follicular  colloid  was  somewhat  increased.  Recent  ex- 
periments on  the  hyperplastic  glands  of  the  opposum  have  amply 
confirmed  these  results;  iodine  administered  daily  to  the  animal 
with  a  hyperplastic  gland  produces  a  gradual  involution  marked 
by  the  slow  accumulation  in  the  cells  of  colloid  droplets,  usually 
a  single  drop  to  a  cell,  and  the  ultimate  discharge  of  these  into 
the  newly  formed  lumen.  We  may  consider  it  proven  therefore 
that  the  production  of  colloid  under  certain  special  conditions  is 
by  this  method. 


44  R.    R.    BENSLEY 

This  conclusion  raises  the  question  whether  the  production  of 
colloid  under  normal  conditions  of  functioning  is  by  the  same 
method,  and,  if  so,  what  are  the  implications  of  this  fact  from  the 
standpoint  of  secretory  rate? 

Hurthle  claimed  that  the  formation  of  colloid  droplets  in  the 
epithelial  cells  of  the  thyroid  gland  was  one  of  the  ways  of  for- 
mation of  colloid  and  that  their  presence  was  an  indication  of 
accelerated  thyroid  activity.  Langendorff  pointed  out  that  they 
were  extremely  rare,  and  therefore  could  not  have  the  secretory 
importance  claimed  by  Hurthle.  That  Langendorff's  contention 
in  this  respect  is  correct  will  readily  be  admitted.  Indeed,  one 
may  search  complete  series  of  sections  of  small  thyroid  glands, 
and  thousands  of  sections  of  larger  ones  without  finding  in  them 
a  single  droplet  of  intracellular  colloid.  In  six  thyroid  glands  of 
man  obtained  at  autopsies  on  executed  criminals,  and  examined 
by  the  writer,  only  one  contained  epithelial  cells  with  colloid 
droplets  in  them.  In  pathological  glands  from  cases  of  exoph- 
thalmic goiter,  simple  colloid  goiter,  and  colloid  adenoma,  on  the 
other  hand,  they  occurred  with  variable  frequency.  In  the  one 
normal  gland  that  contained  them  the  colloid  drops  occurred  with 
great  frequency.  For  the  most  part  they  were  placed  not  at  the 
free  margin  of  the  cell,  but  deep  in  the  protoplasm,  often  along- 
side of  the  nucleus,  and  in  many  cases  several  drops  formed  a  row 
extending  from  this  deeper  location  to  the  free  border.  In  many 
follicles,  however,  the  colloid  droplets  occupied  the  tips  of  the 
epithelial  cells,  and  in  others  the  colloid  masses  inside  the  follicle 
could  be  seen  to  be  made  up  of  a  cluster  of  small  droplets,  ap- 
parently derived  from  different  cells,  which  had  failed  to  fuse 
with  one  another  inside  of  the  follicle.  This  gland  also  con- 
tained an  unusual  number  of  colloid  cells  of  Langendorff. 

The  obvious  participation  of  these  intracellular  colloid  drop- 
lets in  the  replenishment  of  the  intrafollicular  colloid,  on  the 
one  hand,  and  the  slowness  of  this  process  demonstrated  by  expe- 
riment, and  the  rarity  of  the  occurrence  of  such  droplets  under 
normal  conditions,  on  the  other  hand,  suggest  the  following  pos- 
sibilities, which,  however,  are  not,  as  will  appear  more  clearly 
later,  mutually  exclusive:  (1)  the  formation  of  colloid  is  an  inter- 


NORMAL  MODE  OF  SECRETION  IN  THYROID  GLAND  45 

mittent  function  of  the  thyroid  cells;  (2)  there  are  other,  at  pres- 
ent unknown  mechanisms  for  the  formation  of  colloid,  correlated 
with  droplet  formation,  but  able  to  proceed  without  it;  (3)  the 
secretion  of  colloid  into  the  gland  lumen  is  an  accessory  and  not 
the  primary  function  of  the  epithelial  cells  of  the  gland. 

For  the  reasons  mentioned  above,  it  is  apparent  that  the  for- 
mation of  colloid  droplets  in  the  cell,  at  least,  is  an  intermittent 
function.  It  is  possible,  however,  that  in  addition  to  this  mode 
of  formation  of  colloid  there  is  a  slow  and  continuous  production 
of  colloid  at  the  free  margin  of  the  cell  unaccompanied  by  the 
formation  of  visible  secretion  antecedents  in  the  cytoplasm,  and 
it  may  be  that  this  latter  is  the  main  method  of  production  of 
intrafollicular  colloid,  the  droplet  method  representing  some 
upset  of  secretory  equilibrium  which  results  in  the  accumulation 
of  the  product  of  secretion  in  the  cell,  instead  of  the  lumen. 
That  this  hypothetical  upset  is  of  the  nature  of  an  acceleration 
of  the  secretory  rate  is,  however,  excluded  by  the  fact  that  we 
often  see  the  droplet  formation  in  the  greatest  abundance  in 
adenomata  the  stroma  of  which  is  in  an  advanced  state  of  hyaline 
degeneration,  and  in  which,  therefore,  there  can  be  no  question  of 
accelerated  secretion  rate.  Histologically  considered  such  a  con- 
ception of  the  process  of  secretion  in  the  thyroid  gland  must 
remain  hypothetical,  since  it  is  incapable  of  objective  proof. 

The  third  possibility,  namely,  that  the  secretion  of  colloid  in- 
to the  gland  lumen  is  an  accessory  and  not  the  primary  function 
of  the  epithelial  cells,  though  correlated  intimately  with  this 
primary  function,  would,  if  established,  explain  and  include  all 
of  the  facts.  Such  a  theory  to  be  accepted,  must  account  for  the 
irregular  occurrence  of  droplets,  for  their  formation  in  the  inte- 
rior of  the  cell  rather  than  on  either  of  the  free  surfaces,  and 
for  their  increase  under  iodin  or  thyreoglobulin  administration, 
and  under  the  experimental  conditions  of  Hurthle.  It  involves 
the  assumption  of  a  more  remote  antecedent  of  the  secretion  than 
the  colloid  droplets  of  Hurthle. 

In  my  studies  of  the  thyroid  glands  of  various  mammals,  I 
have  been  struck  with  the  frequent  occurrence,  particularly  in 
the  cat,  dog  and  opposum,  of  vacuoles  with  unstainable  con- 


46  R.    R.    BENSLEY 

tents,  rather  irregular  in  shape,  occurring  in  the  base  of  the  cell, 
and  with  the  frequent  occurrence  in  the  cells  of  hyperplastic 
human  glands  from  cases  of  true  exophthalmic  goiter  of  droplets 
of  material  staining  like  colloid  located  similarly  in  the  extreme 
bases  of  the  cell  near  the  capillary  net.  Ferguson  ('11),  also, 
has  described  the  occasional  occurrence  in  the  thyroid  gland  of 
elasmobranch  fishes  of  cells  presenting  in  their  basal  ends  a 
ragged  and  rodded  appearance  which  he  interprets  as  due  to 
secretion  storage  for  direct  export  to  the  vascular  channels. 

A  group  of  opossums  kept  under  observation  under  various 
experimental  conditions  during  the  past  winter  have  furnished 
material  in  which,  by  reason  of  the  fact  that  these  vacuolar  sub- 
stances in  the  cell  were  unusually  increased  in  amount,  it  was 
possible  to  study  their  variation  and  to  develop  a  technique  for 
staining  of  their  contents.  One  group  of  these  animals  was  kept 
for  a  period  of  three  weeks  on  a  dietary  consisting  of  beef,  bread 
and  fat,  egg,  bread  and  fat,  or  cheese;  bread  and  fat,  just  suffi- 
cient to  maintain  constant  weight.  In  another  group  the  diet 
was  so  regulated  that  with  constant  bread  and  fat  content  there 
was  a  progressive  increment  of  meat  fed  to  the  successive  mem- 
bers of  the  series.  In  all  of  the  animals  thus  kept  on  a  controlled 
diet,  the  thyroid  cells  contained  such  basal  vacuoles,  and  in  two 
of  the  animals  of  the  second  series,  namely  those  which  received, 
respectively,  twice  and  two  and  a  half  times  the  normal  meat 
ration,  the  material  of  this  sort  comprised  fully  half  of  the  cell 
contents. 

The  fixation  of  the  material  is  of  considerable  importance  in 
the  study  of  these  vacuolar  substances,  because  the  contents 
are  so  dilute  that  they  may  be  precipitated  in  an  invisible  form 
on  the  protoplasmic  strands  which  wall  the  vacuoles.  Formalin 
zenker,  however,  was  found  to  precipitate  the  contents  in  the  form 
of  a  thin  gel  sometimes  filling  completely  the  space  of  the  vacu- 
oles, sometimes  containing  small  vacuoles  from  contraction  in 
fixation.  Staining  however  was  difficult,  because  the  material 
stained  with  the  usual  dyes  in  the  same  way  as  the  protoplasm. 
With  Mallory's  connective  tissue  stain,  however,  it  could  be  seen 
that  the  vacuoles  had  vaguely  staining  contents,  but,  since  the 


NORMAL  MODE  OF  SECRETION  IN  THYROID  GLAND      47 

protoplasm  also  stained  bluish  after  formalin  zenker  fixation,  it 
was  difficult  to  define  accurately  the  limits  of  the  vacuoles,  and 
after  fixation  in  ordinary  Zenker's  fluid  the  vacuoles  did  not 
stain  at  all.  Accordingly  the  indication  was  to  find  some  stable 
stain  which  would  stain  the  protoplasm  diffusely,  and  then  to 
stain  the  secretion  a  contrast  color.  For  this  puropse  brasilin 
in  phosphotungstic  acid  solution  was  found  to  be  effective.  The 
solution  is  prepared  as  follows: 

Phosphotungstic  acid 1.0    g. 

Distilled  water 100.0    cc. 

Brasilin 0.05  g. 

The  brasilin  is  first  dissolved  in  a  small  quantity  of  distilled 
water  by  the  aid  of  heat  and  added  to  the  phosphotungstic  acid 
solution.  Ripening  may  be  accelerated  by  the  addition  of  0.4 
cc.  of  hydrogen  peroxide,  or  of  a  few  drops  of  a  solution  of  soluble 
molbydic  acid.  The  solution  deteriorates  with  age  and  should 
not  be  used  after  three  days. 

Sections  of  thyroid  glands  which  have  been  fixed  in  formalin 
zenker,  fastened  to  slides  by  the  water  method  (if  albumen  is 
used  it  should  be  very  small  in  amount)  are  passed  through 
toluol,  absolute  alcohol,  to  water,  iodised,  and  placed  in  the 
staining  solution  from  one  to  several  hours.  The  sections  are 
then  washed  in  water  and  placed  for  one  to  five  minutes  in  the 
following  solution: 

Phosphomolybdic  acid 1.0  g. 

Wasserblau 0.2  g. 

Water 100.0  cc. 

Then  wash  rapidly  in  water,  dehydrate  in  absolute  alcohol,  clear 
in  toluol,  and  mount  in  balsam. 

In  the  preparations  so  stained  with  brasilin  and  wasserblau 
the  cytoplasm  stains  pink  to  lilac,  the  nuclear  chromatin,  deep 
red,  and  the  contents  of  the  vacuoles  sky  blue,  as  shown  in  Fig. 
1.  The  colloid  droplets  of  Hiirthle  stain  deep  blue  or  deep  red 
according  to  the  concentration  of  the  gel  which  composes  them, 
which  determines  the  diffusion  rate  of  the  dyes  employed. 


48  R.    R.    BENSLEY 

It  must  not  be  supposed  that  the  technical  difficulties  of  study- 
ing tftis  intracellular  product  are  wholly  overcome  by  the 
method  just  described.  When  the  material  is  large  in  amount 
the  method  is  very  satisfactory,  but  the  intensity  of  the  proto- 
plasmic staining  is  not  sufficient  to  define  the  material  sharply 
when  it  is  small  in  amount.  Under  these  circumstances,  a  brief 
mordanting  of  the  section,  before  staining,  in  a  fresh  solution  of 
ammonium  stannic  chloride  will  improve  the  contrast  staining, 
but  will  detract  greatly  from  the  transparency  and  beauty  of  the 
preparation. 

The  examination  of  the  sections  of  the  experimental  series  re- 
ferred to  above,  and  of  a  number  of  normal  glands  from  animals 
killed  as  soon  as  obtained,  reveals  the  presence  in  all,  although 
in  highly  variable  amounts  in  the  individual  members  of  the 
series,  of  a  new  secretion  antecedent.  This  substance  is  in  the 
form  of  vacuoles,  occurring  exclusively  in  the  outer  pole  of  the 
cell,  which  contain  a  dilute  solution  similar  in  its  properties  to  the 
colloid  of  the  follicular  lumen,  differing  from  the  latter  only  in 
density.  There  are  even  in  this  substance  clear  vacuoles  due  to 
shrinkage  in  fixation,  like  those  seen  in  the  colloid  of  the  lumen. 

In  two  members  of  the  experimental  series,  this  substance  is 
present  in  such  amount  that  it  fills  quite  half  the  cell.  In  these 
cases  the  cell  presents  an  appearance  comparable  to  that  of  the 
secreting  cells  of  an  exocrine  gland  like  the  pancreas,  with  the 
exception  that  the  hylogens  are  in  dilute  solution  in  fairly  large 
vacuoles  instead  of  in  the  form  of  granules,  and  they  are  in  the 
basal  end  of  the  cell  instead  of  the  free  end.  In  other  words 
these  cells  exhibit  the  ordinary  picture  of  a  secreting  cell  with 
stored  secretion  antecedents,  but  with  reversed  polarity. 

Figure  1  shows  an  acinus  from  one  of  these  glands.  The  cells 
are  cylindrical  in  shape  with  a  spherical  nucleus  placed  rather 
nearer  to  the  free  end  of  the  cell  than  to  the  base.  The  base  of 
the  cell  is  filled  with  sky-blue  stained  material  contained  in 
vacuoles  separated  from  one  another  by  thin  sheets  of  cytoplasm 
containing  mitochondrial  filaments.  The  free  pole  of  the  cell 
directed  towards  the  lumen  is  finely  granular  and  stained  a 
bluish-pink  color.  It  contains  none  of  the  blue  staining  vacuolar 


NORMAL  MODE  OF  SECRETION  IN  THYROID  GLAND      49 

material,  and  consists  solely  of  cytoplasm  containing  crowded 
rmtochondrial  filaments.  In  two  cells  of  this  figure  small  glob- 
ules of  colloid  may  be  seen,  in  one  case  alongside  of  the  nucleus, 
and  in  another  in  the  apical  cytoplasm. 

In  some  of  the  thyroid  glands  obtained  from  opossums  recently 
captured,  consisting  of  fairly  large  follicles  well  filled  with  col- 
loid, the  epithelial  cells  appeared  uniformly  vacuolated,  but  when 
the  preparations  were  stained  with.brasilin  and  wasserblau  the 
vacuoles  in  the  outer  ends  of  the  cells  were  found  to  be  filled  with 
blue  staining  material,  those  in  the  inner  ends  with  unstainable 
material. 

Three  possible  interpretations  of  the  presence  of  this  material 
suggest  themselves:  first,  that  it  is  a  pathological  product  rep- 
resenting cytoplasmic  degeneration,  or  imbibed  serous  fluid,  or 
simple  edema;  second;  that  it  is  colloid  in  process  of  resorption 
by  a  transcellular  route;  third,  that  it  is  a  true  secretion  ante- 
cedent representing  material  formed  in  the  base  of  the  cell  for 
the  purpose  of  direct  transport  into  the  vascular  channels. 

The  fact  that  every  cell  of  the  gland  contains  the  material, 
that  it  is  present  in  some  degree  in  all  opossum  thyroid  glands, 
and  that  there  are  no  other  evidences  of  degeneration,  such  as 
changes  in  the  nucleus  or  in  the  mitochondria,  or  in  the  intra- 
lobular  connective  tissue  excludes  the  first  possibility  from  con- 
sideration. 

Opposed  to  the  second  of  these  hypotheses  is  the  fact  that 
only  very  exceptionally  is  this  material  found  in  the  pole  of 
the  cell  in  contact  with  the  follicular  content,  and  then  only  when 
the  cell  is  so  loaded  with  the  secretion  that  it  is  comparable  in 
appearance  to  a  parotid  gland  cell  filled  with  zymogen  granules. 
It  might  be  possible  to  assume  that  the  droplets  of  colloid  occa- 
sionally seen  in  the  cell  are  on  the  way  out  rather  than  proceeding 
towards  the  lumen.  The  evidence  from  the  glands  which  are  be- 
ing reverted  by  iodine  is,  however,  strictly  opposed  to  this  hy- 
pothesis, since  a  progressive  increase  in  colloid  in  these  cases  has 
been  demonstrated  in  an  experimental  series  taken  at  different 
intervals  of  time  and  this  increase  is  associated  directly  with 
colloid  droplet  formation  and  extrusion  into  the  lumen.  It  is 


50  R.    R.    BENSLEY 

conceivable,  of  course  that  the  droplets  of  colloid  are  of  two  sorts, 
those  destined  for  the  follicular  content,  and  those  on  their  way 
to  the  base  of  the  cell  for  secretion  into  the  vascular  channels  of 
the  gland.  Opposed  to  this  assumption  is  the  fact  that  in  entire 
glands,  in  the  cells  of  which  there  is  an  abundance  of  the  basal 
vacuolar  substance  there  may  be  found  not  a  single  droplet  of 
colloid  of  the  dense  type,  and  that  the  free  poles  of  all  the 
cells  may  be  wholly  free  from  products  of  secretion  except  where 
the  crystals,  of  protein  nature,  demonstrated  in  a  former  article, 
project  into  this  pole.  Furthermore,  in  hyperplasia  of  long 
standing,  in  which  there  is  practically  no  intrafollicular  colloid, 
a  large  content  of  the  new  secretion  in  the  form  of  small  vacuoles 
distributed  throughout  the  outer  pole  of  the  cell,  may  be  present. 

We  are  therefore  forced  to  accept  the  third  hypothesis  which, 
physiologically  considered,  is  the  more  attractive,  inasmuch  as  it 
permits  of  harmonizing  the  various  facts  under  a  single  hypothe- 
sis, namely  that  the  secretion  collected  in  the  outer  pole  of  the 
thyroid  cell  is  destined  to  direct  transport  into  the  vascular  chan- 
nels, and  that  the  thyroid  cell  represents  a  true  reversal  of 
polarity  in  accord  with  its  endocrine  function. 

In  addition  to  the  facts  mentioned  above  which  point  strongly 
to  the  correctness  of  this  hypothesis,  it  may  be  pointed  out 
that  in  exocrine  glands  fat  droplets  which  are  deposited  in  the 
secreting  cells  practically  always  make  their  appearance  at  the 
anti-secretory  pole  of  the  cell;  this  is  the  case  in  the  pancreatic 
cells  and  in  the  chief  cells  of  the  gastric  glands.  The  location 
of  the  fat  deposits  in  the  thyroid  gland  also  is  at  the  pole  which 
according  to  the  hypothesis  here  supported  is  the  anti-secre- 
tory pole  of  the  cell,  namely  the  free  end  of  the  cell  next  the 
colloid. 

We  may  assume  therefore  that  the  thyroid  gland  as  all  physio- 
logical and  clinical  experience  indicates,  prepares  and  secretes 
into  the  vascular  channels  of  the  gland  a  secretion,  and  that  this 
secretion  is  formed  in  the  outer  pole  of  the  cell,  and  excreted  from 
it  directly  under  normal  conditions  of  functioning  without  passing 
by  the  indirect  route  through  the  follicular  cavity. 


NORMAL  MODE  OF  SECRETION  IN  THYROID  GLAND  51 

It  is  necessary,  however,  under  this  hypothesis  to  explain  the 
occurrence  of  intrafollicular  colloid,  and  its  variability  in  differ- 
ent members  of  a  species  and  under  different  experimental  con- 
ditions. In  my  recent  studies  on  the  changes  in  the  hyperplastic 
gland  of  the  opossum  and  its  changes  under  domestication,  I  have 
shown  that  all  the  scored  colloid  may  be  withdrawn  from  the 
gland  in  a  short  period,  and  that  the  gland  will  maintain  for  a 
period  of  several  months  a  condition  in  which  little  visible  colloid 
is  present  in  the  gland,  but,  as  Marine  previously  demonstrated 
in  the  dog,  if  iodine  be  administered  the  vesicles  are  reformed ,  and 
filled  with  dense  colloid.  In  the  opossum  this  colloid  makes  its 
appearance  first  deep  in  the  thyroid  cells,  but  migrates  to  the 
free  surface  and  is  there  discharged  into  the  lumen.  I  have  also 
found  in  the  study  of  many  glands  from  cases  of  Basedow's  dis- 
ease that  the  few  colloid  droplets  which  are  present  are  very  fre- 
quently found  at  the  level  of  the  nucleus,  or  even  in  the  base  of 
the  cell.  These  facts  indicate  that  in  addition  to  the  direct 
mode  of  secretion  there  is  an  indirect  mode,  which  consists  in  the 
condensation  of  the  secretion  into  the  form  of  droplets  having  a 
high  content  of  solids,  and  the  extrusion  of  these  droplets  into  the 
follicular  cavity.  These  droplets  are  formed  in  the  same  zone  of 
the  cell  as  that  in  which  the  primary  or  direct  secretion  is  formed, 
and  it  is  probable  that  they  are  formed  at  the  expense  of  the 
latter. 

The  readiness  with  which  the  thyroid  gland  undergoes  hyper- 
plastic  change,  its  responsiveness  to  iodine  administration,  as 
demonstrated  by  Marine  and  his  co-workers,  the  ease  by  which 
it  may  be  modified  structurally  by  dietary  conditions  as  shown  by 
the  work  of  Reid  Hunt  ('11),  Marine,  Chalmers  Watson  ('07), 
Tanberg  ('00),  and  Missiroli  ('10)  and  confirmed  by  my  recent 
studies  on  the  relation  of  diet  to  hyperplasia  in  opossums  under 
domestication,  confirm  the  conclusion  that  there  is  a  delicate  ad- 
justment between  the  functioning  of  the  thyroid  gland  and  general 
body  conditions,  though  at  present  we  do  not  know  the  means 
by  which  this  adjustment  is  mediated.  This  being  the  case  it 
may  be  assumed  that  only  when  this  adjustment  is  disturbed  so 
that  the  rate  of  secretion  is  in  excess  of  body  needs,  the  indirect 


52  R.    R.    BENSLEY 

mode  of  secretion  comes  in,  and  the  product  of  secretion  is  con- 
densed and  stored  in  the  intrafollicular  cavity.  It  is  conceiv- 
able, of  course,  that  other  factors  than  excess  of  production  over 
functional  needs  might  bring  about  this  result,  as,  for  example, 
an  agent  inhibiting  direct  export  from  the  cell,  or  mechanical  in- 
terference with  the  outflow  from  the  cells,  The  latter  influence 
is  well  illustrated  in  the  colloid  adenomata  where,  notwithstanding 
the  fact  that  the  stroma  may  be  hyaline,  the  cells  contain  abun- 
dant colloid  spherules,  and  thus  give,  according  to  the  old  criterion 
of  Hiirthle,  the  impression  of  high  secretory  activity.  According 
to  my  hypothesis  of  thyroid  secretion,  this  condition  would  rep- 
resent a  slow  secretory  activity  of  the  epithelium  of  the  tumor, 
all  of  the  energy  of  which  is,  however,  devoted  to  storage,  since 
direct  export  by  way  of  the  vascular  channels  is  impossible. 
This  may  explain  the  difference  noted  by  Marine  between  the 
hyperplastic  gland  and  the  adenoma  as  to  susceptibility  to  influ- 
ence by  iodine.  The  hyperplastic  gland,  whether  its  activity  be 
high  or  low,  is  exporting  its  product  directly.  Iodine  whether 
by  accelerating  the  activity  of  the  gland  and  so  producing  a  con- 
dition of  physiological  saturation  with  thyroid  products,  or  by 
actually  inhibiting  the  export  of  material  from  the  cell,  causes  the 
cell  to  reverse  its  processes,  and  store  it  in  the  follicular  cavities. 
The  adenoma  is  already  storing  all  of  the  product  which  the 
vascular  conditions  and  its  specific  cell  equilibrium  permit  it  to 
form,  and  so  the  process  can  not  be  influenced  by  iodine. 

According  to  this  conception  of  thyroid  secretion  the  colloid 
in  the  thyroid  vesicles  is  per  se  no  measure  of  the  activity  of 
the  gland  at  the  moment  of  observation,  though  its  consistence 
and  its  qualities  may  offer  valuable  indications  of  the  capacity 
of  the  thyroid  cell  for  normal  storage.  The  colloid  in  fact  may 
be  the  product  of  a  storage  phase  which  preceded  the  exafnina- 
tion  of  the  gland  by  a  considerable  period  of  time,  since  it  is 
necessary  to  assume  the  resorption  of  this  material  only  under  the 
conditions  where  the  normal  direct  secretory  activity  of  the,  gland 
is  insufficient  to  meet  the  functional  demands.  Accordingly, 
also,  lack  of  colloid  in  the  gland  does  not  necessarily  mean  de- 
pression of  the  gland  activity  below  the  normal  rate  at  the  time 
of  observation,  though  it  probably  does  mean  that  there  is  such  a 


NTORMAL  MODE  OF  SECRETION  IN  THYROID  GLAND  53 

depression  of  physiological  efficiency  or  has  been  at  some  pre- 
vious time,  and  either  that  the  gland  has  not  risen  above  the 
level  of  secretory  rate  needed  for  direct  export,  or  that  there  has 
been  a  failure  of  the  normal  mechanism  of  regulation.  My  ob- 
servations on  the  hyperplastic  glands  of  opossums  by  means 
of  the  methods  described  in  this  paper  have  shown  that  hyper- 
plastic  glands  which  appear  almost  identical  histologically  may 
yet  differ  markedly  in  the  amount  of  these  intracellular  secre- 
tion antecedents  which  they  contain,  and  thus,  probably,  in 
secretory  potential. 

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THK  AMERICAN  JOURNAL  OF  ANATOMY,  VOL.  19,  NO.  1 


54  R.    R.    BENSLEY 


EXPLANATION  OF  PLATE 

1  A  group  of  follicles  from  the  thyroid  gland  of  the  opossum,  fixed  in  forma- 
lin zenker,  stained  with  brasilin-wasserblau.  X  1050.  In  the  outer  poles  of  the 
cells  a  material  differentially  stained,  similar  to  the  intrafollicular  colloid.  In 
two  cells  droplets  of  colloid  destined  for  storage. 


NORMAL  MODE  OF  SECRETION  IN  THYROID  GLAND 

R.  R.  BEN8LBY 


PLATE  1 


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